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Up to now, excretion of CGA after coffee consumption was observed by monitoring renal excretion, although an excretion via the enterohepatic circulation is theoretically possible as well.

The overall renal excretion of an oral ingested drug such as CGA can be presumed to give evidence of its bioavailability as the concentration of a drug at the site of drug action cannot be directly measured readily (Chereson, 1996). A comparative consideration of data on renal excretion after CGA or HCA consumption is hindered, because factors which may influence the bioavailability differ in the considered studies (see Table 2-13). Different doses and dosage forms with partially different CGA and HCA compositions were administered and additionally different analytical methods were used for detection of compounds in urine.

More specifically, Olthof and coworkers observed a different renal excretion after the consumption of CA or 5-CQA as pure compounds dissolved in hot water by ileostomists (Olthof et al., 2001). This finding provides evidence for a different absorption and excretion behavior according to the CGA or HCA composition of the consumed food. The consumed artichoke leaf extract (containing CGA and flavanoids) (Wittemer et al., 2005), the polyphenol rich extract (Ito et al., 2005) and the high bran cereal breakfast (Kern et al., 2003a) showed completely different polyphenol profiles to that of coffee, so the comparability of the described studies is less. Studies which determined the renal excretion of CGA after coffee consumption are different in administered dose and dosage forms, too. Farah and coworkers administered an encapsulated green coffee extract, which had to be disintegrated in the GIT before absorption (Farah et al., 2008), Rechner administered 6 cups of coffee within 8 hours (Rechner et al., 2001) and Stalmach administered a single dose of coffee to healthy (with colon) and ileostomists (without colon) (Stalmach et al., 2009; Stalmach et al., 2010) see Table 2-13.

Thus, it is not possible to draw a conclusion based on available literature data, whether the different doses and dosage forms are affecting the bioavailability.

However, Wittemer et al. reported a renal excretion of about 4% of the ingested CGA after the consumption of two artichoke leaf extracts with two different CA equivalent amounts indicating a dose independent excretion (Wittemer et al., 2005).

Additionally, the analytical methodology influences the outcome, as shown by the group of Stalmach (Stalmach et al., 2009; Stalmach et al., 2010). They used an analytical method without enzyme hydrolysis of CGA metabolites and detected a multiple level of renal excretion compared to others (see Table 2-13). The metabolites were measured directly by Stalmach et al. with a high selective HPLC-MS system in the multiple ion mode. Other groups reported significantly lower amounts excreted renally by using enzymatic hydrolysis of metabolites. These groups measured the aglyca by HPLC-DAD (Olthof et al., 2001; Rechner et al., 2001;

Kern et al., 2003a; Farah et al., 2008), LC-MS/MS (Ito et al., 2005), or HPLC coulometric array detection (Wittemer et al., 2005).

On the one hand an intervention study with different CGA doses in a single food matrix is required to investigate the influence of different doses on CGA bioavailability. On the other hand an individual dose and different matrices are required to answer the influence of different food matrices on bioavailability.

Additionally, those studies have to be performed with a CGA source with a defined CGA and HCA composition and an analytical methodology for the excreted compounds without enzymatic hydrolysis.

Table 2-13: Summary of renal excretion after consumption of chlorogenic or hydroxycinnamic acids (within food matrix) in probands with colon (healthy volunteers). Renal excretion is given in % of ingested dose based on total cinnamates. Comparative consideration of renal excretion between the single studies is limited because of the different consumed doses, different dosage forms and different analytical methods used for measurement (with or without metabolite detection). FA = ferulic acid (7a).

Dose Dosage form Age BMI Subjects Renal excretion Reference

(mg) Matrix Consumption (a) (kg*m^-2) % of dose* extract; 10 g cocoa powder; 18 g grape skin extract; 200 mL

not specified 25 22 5 F 4 M 3.8 ^Ɨ (Ito et al., 2005)

encapsulated, 0.4 g fasted (light meal after 1 h)

22 - 55 n.d. 5 F 5 M 5.5^Ɨ (Farah et al., 2008)

898 coffee 6 cups within 8 hours not specified 31 25 5 M ≈ 5.9^Ɨ (Rechner et al., 2001)

*as total cinnamates; °° CA equivalents; ^Ɨ after enzymatic hydrolysis (without metabolite identification); n.d. = not determined; F = female; M = male

Table 2-14: Summary of renal excretion after consumption of chlorogenic or hydroxycinnamic acids (as pure compound or within food matrix) in probands without a colon (ileostomy volunteers). Renal excretion is given in % of ingested dose based on total cinnamates. Comparative consideration of renal excretion between the single studies is limited because of the different consumed doses, different dosage forms and different analytical methodology used for measurement (with or without metabolite detection).

CA = caffeic acid (5a), 5-CQA = 5-caffeoylquinic acid (1d).

Dose Dosage form Age BMI Subjects Renal excretion Reference

(mg) Matrix Consumption (a) (kg*m^-2) % of dose*

Subjects without colon (ileostomists)

139 coffee 200 mL fasted (light

meal after 3 h)

41 – 54 26 2 F 3 M 8 (Stalmach et al., 2010)

500 CA 200 mL hot water with light

breakfast

63 27 4 F 3 M 10.7^Ɨ (Olthof et al., 2001)

1,000 5-CQA 200 mL hot water with light

breakfast

63 27 4 F 3 M 0.6^Ɨ (Olthof et al., 2001)

* as total cinnamates; °° CA equivalents; ^Ɨ after enzymatic hydrolysis (without metabolite identification); n.d. = not determined; F = female;

M = male

3 Aims

Several previous studies on CGA oral bioavailability revealed contradictory data (Olthof et al., 2001; Kahle et al., 2007; Stalmach et al., 2010; Hagl et al., 2011;

Williamson et al., 2011). Different factors can have an influence on the oral bioavailability of CGA, such as the dosage form, dose, proband’s physiology or the molecular properties of the drug (Chereson, 1996; Lipinski et al., 1997; Dietrich et al., 2003; Williamson et al., 2011; El-Kattan and Varma, 2012). As different studies with healthy (see Table 2-10, State of knowledge) or ileostomy volunteers (see Table 2-9 and Table 2-14, State of knowledge) showed methodological differences in their analytical strategies (sample preparation with or without enzymatic cleavage of CGA conjugates), dosage form (such as different CGA composition from different food matrix) or dose, the influence of a single parameter on bioavailability could not be observed.

As coffee is the richest source of CGA in the western diet and a common coffee beverage (200 mL) serves between 70 and 350 mg CGA (Clifford, 1999; Farah and Donangelo, 2006) the main objective of this thesis was to monitor the effects of CGA and QA dose from coffee on their oral bioavailability. Especially an influence of ingested dose on the site where absorption occurs and metabolization was here in a closer focus. Therefore, ileostomy volunteers consumed three different doses of CGA and QA by coffee brew and the effect of dose was determined on: ileal excretion, plasma concentration, and renal excretion of QA, CGA and its metabolites. This dose-response study was performed in cooperation with the Department of Medicine II, Gastroenterology, University of Wuerzburg, Germany.

Furthermore, the CGA composition of coffee beverages or other food matrices containing CGA vary (Farah and Donangelo, 2006; Neveu et al., 2010). As varying CGA compositions show different physico-chemical properties this could also have an influence on CGA bioavailability. However, this has not been sufficiently investigated for coffee CGA up until now. Especially, the effect of the physico-chemical properties of CGA from coffee on their individual absorption rates has not been investigated. Here, the objective was to determine effects of the physico-chemical properties on absorption of CGA and QA. This was performed ex

vivo using the Ussing chamber model, with pig jejunum and individual compounds in physiological concentrations.

4 Results

In this work we investigated the bioavailability of coffee CGA and QA in the upper parts of the GIT. More specifically, we observed an influence of the consumption of different coffee doses on CGA and QA bioavailability in ileostomists and furthermore the influence of the chemical structure of CGA and QA on the bioavailability of those molecules in the pig jejunal mucosa model.

Therefore, we established on the one hand an ex vivo absorption experiment with pig jejunal mucosa in the Ussing chamber using CGA and QA present in coffee to observe effects of the chemical structure on their bioavailability. For this, these molecules were applied in physiological concentrations (20 µM) and their total absorbed amounts were determined (luminal to serosal).

Moreover, the mechanism of absorption was elucidated for 5-CQA, which is the dominating CGA in coffee. In order to differentiate between diffusion and active transport the absorption rates (flux) were measured at concentrations from 20 µM up to 3,500 µM (luminal to serosal). Secretion of CGA (serosal to luminal direction) were measured with 5-CQA at 20 µM with and without co-incubation of the metabolic inhibitor NaN3 (10mM). Furthermore, active efflux transporters were detected in the pig jejunal mucosa by Western blot analysis.

In order to investigate the influence of different CGA doses on CGA bioavailability we performed a randomized, double-blinded and dose-response intervention study. On three separate days ileostomists consumed decaffeinated coffee with three different doses of CGA (HIGH 4,525 µmol; MEDIUM 2,219 µmol; LOW 1,053 µmol) and QA (HIGH 2,546 µmol, MEDIUM 1,373 µmol, LOW 695 µmol). The influence of the different consumed doses on total absorbed amounts (renal excretion), colonic availability (ileal excretion), metabolization and plasma concentrations was observed.

Further, the stability of the mentioned coffee molecules in ileal fluid was investigated to observe any possible degradation during sample collection.